Proteins Folding: The Big Questions

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Proteins Folding The Big Questions

• Can 3-D structure be predicted from sequence?
• Can sequence for a specific 3-D structure be
  predicted?

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Struthers, Cheng, Imperiali, Science 1996, 271,
342
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Bryson et al., Science, 1995, 270, 935
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Struthers, Cheng, Imperiali, Science 1996, 271,
342
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Dahiyat, Mayo, Science 1997, 278, 82
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Dahiyat, Mayo, Science 1997, 278, 82
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Dahiyat, Mayo, Science 1997, 278, 82
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Design of a hyperstable protein
Malakauskas, Mayo, Nature Struct. Biol. 1998, 5,
470
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Malakauskas, Mayo, Nature Struct. Biol. 1998, 5,
470
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Malakauskas, Mayo, Nature Struct. Biol. 1998, 5,
470
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Design of enzymatic activity The Holy Grail of
de novo design

• A story of computation vs. phage display
• Early days
• A couple cautionary tales
• Recent success!

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Science 1986, 234, 1570
Rate enhancement vs uncatalyzed 770 M/M
kinetics inhibited by hapten to IgG
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Catalytic antibodies raised against reaction
transition states

• Typically show rate enhacements that are 104 vs
  uncatalyzed rates (range 103-106)
• Synthesis of TS analog can be challenging
  usually compromises in the structure of the
  analog vs. TS are made

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Oxaldie
Rate enhancement vs uncatalyzed 104 Natural
enzyme vs uncatalyzed 108 Helical vs.
non-helical mutant 10
Johnsson et al, Nature 1993, 365, 530
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Wagner, Lerner, Barbas, Science 1995, 270, 1797
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Wagner et al., Science 1995, 270, 1797
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Rate enhancement vs uncatalyzed 109 FBP aldolase
vs uncatalyzed 4 1012
Wagner, Lerner, Barbas, Science 1995, 270, 1797
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Tanaka, Barbas, J. Immunol. Meth. 2002, 269, 67
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Prodrug activation site-selective activation
of chemotherapeutics
Tanaka, Barbas, J. Immunol. Meth. 2002, 269, 67
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Rate enhancement vs uncatalyzed 1900 But with KM
1.8 mM
Can greater specificity be engineered into this
catalytic peptide? Add 12mer peptide that
binds fluorescein specifically
Tanaka, Barbas, JACS. 2002, 124, 3510
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Can an existing protein scaffold be engineered to
include catalytic residues (in this case a
Ser-His-Asp catalytic triad)? Step 1 make
several active site mutants of cyclophilin to
introduce a Ser Step 2 add His and Asp
mutations to best Ser mutant
Quéméneur et al, Nature 1998, 391, 301
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and appears to depend on a deprotonated His for
optimum activity. It is also inhibited by
nucleophile-specific chemical agents
The new protease activity is specific for
cleavage after Pro
Quéméneur et al, Nature 1998, 391, 301
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Almost 109 rate enhancement for the triple mutant.
Quéméneur et al, Nature 1998, 391, 301
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• PRAI and IGPS catalyze consecutive reactions in
  the Trp biosynthetic pathway. Both are TIM
  barrel proteins.
• TIM barrels
• Common 250 aa structural motif (10 of all
  characterized proteins)
• S binding within barrel catalytic residues tend
  to be in loops outside barrel

Goal convert IGPS to have PRAI activity
Altamirano et al, Nature 2000, 403, 617
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IGPS green PRAI blue
Altamirano et al, Nature 2000, 403, 617
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Step 1 delete 48 residues from N-term of
IGPS Result destabilized, insoluble protein
inactive upon refolding Step 2 create library
targeting the ?1?1 loop for replacement by GKXX
through GKXXXXX Result protein soluble but prone
to aggregation binds S but is inactive upon
refolding Step 3 introduce catalytic Asp and
GXGGXGQ into the ?6?6 loop Result from a randomly
picked clone protein soluble CD spectrum like
TIM barrel binds S but is inactive
Altamirano et al, Nature 2000, 403, 617
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Step 4 Screen random library from Step 3 in
complementation assay Result roughly 500 of
30,000 clones grew when supplemented with low Trp
concentration Step 5 Performed DNA shuffling of
putative PRAI genes recovered in Step 4 and
performed complementation screen Result enzyme
that can apparently complement PRAI(-) E. coli
strain.
Altamirano et al, Nature 2000, 403, 617
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Complementation of PRAI-deficient E. coli strain
(doesnt grow in absence of added Trp)
() Trp
(-) Trp
minimal medium
(-) Trp
() Trp
Rich medium
() Trp
(-) Trp
() Trp
360 colonies can grow on (-) Trp
Altamirano et al, Nature 2000, 403, 617
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28 sequence identity to PRAI 90 sequence
identity to IGPS
Altamirano et al, Nature 2000, 403, 617
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Altamirano et al, Nature 2000, 403, 617
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Dwyer, Looger, Hellinga, Science 2004, 304, 1967
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Dwyer, Looger, Hellinga, Science 2004, 304, 1967
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Dwyer, Looger, Hellinga, Science 2004, 304, 1967
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Dwyer, Looger, Hellinga, Science 2004, 304, 1967
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Dwyer, Looger, Hellinga, Science 2004, 304, 1967
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Design of a Novel Globular Protein Fold with
Atomic-Level Accuracy
Kuhlman et al., Science 2003, 302, 1364
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Kuhlman et al., Science 2003, 302, 1364
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Kuhlman et al., Science 2003, 302, 1364
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The design of Top7 shows that globular protein
folds not yet observed in nature not only are
physically possible but can be extremely stable.
The protein therapeutics and molecular machines
of the future should thus not be limited to the
structures sampled by the biological evolutionary
process. The methods used to design Top7 are, in
principle, applicable to any globular protein
structure and open the door to the exploration
and use of a vast new world of protein structures
and architectures.
Kuhlman et al., Science 2003, 302, 1364
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Computational Thermostabilization of an Enzyme
Yeast cytosine deaminase, yCD, (converts cytosine
to uracil) was chosen because 1) its
high-resolution crystal structure is available
2) its catalytic mechanism is well characterized
3) it is thermolabile 4) it has potential use
in antitumor applications As is true of many
commercially useful enzymes, yCD displays
irreversible unfolding behavior at high
temperatures (presumably because of aggregation).
used computational redesign to predict a series
of point mutations in the enzyme core that might
lead to thermostabilization of the enzyme without
losing catalytic efficiency
Korkegian et al., Science 2005, 308, 857
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• All residues within 4Å of active site were held
  constant
• For the mutations allowed at the remaining
  residues (65/153) only Cys was excluded
• 33/65 residues remained wild-type after
  computational design
• 16/65 mutations were on surface and not pursued
• Two clusters and four point mutations emerged
• Cluster 1 mutations (9 total) led to insoluble
  protein
• Cluster 2 (4 total) led to soluble protein and
  high resolution mutagenesis showed that A23L and
  I140L were largely responsible for the
  thermostabilization.
• One point mutant, V108I, also contributed
  significant stability

Korkegian et al., Science 2005, 308, 857
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Mutants still aggregate at higher temps. Each
mutation adds 2 C to stability but, triple
mutant is 10 C more stable than wt
t1/2 _at_ 50 C wt 4 hr double 21 hr
triple 117 hr
Korkegian et al., Science 2005, 308, 857
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Complementation assay on minimal medium lacking
uracil Triple mutant confers rapid growth vs. wt
Korkegian et al., Science 2005, 308, 857
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wt
mutant
Korkegian et al., Science 2005, 308, 857
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The redesigned, mutated residues all appear to
pack more tightly in the enzyme core, with more
surface area in contact with neighboring residues
without altering the nearby side chain rotamers
or backbone conformation. Approximately 70 Å2
of additional buried surface area is incorporated
as a result of the three mutations
Korkegian et al., Science 2005, 308, 857
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De novo design of catalytic proteins
Kaplan, DeGrado, PNAS. 2004, 101, 11566
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The ultimate goal is to design an efficient
catalyst that does not fall into a deep energy
minimum or encounter large energy barriers along
any of these steps. Thus, the immediate goal is
to find the intersection of sequence space that
catalyzes eqn. 13 in Scheme 1.
The catalytic peptides were designed by varying
the sequence of DFtetAaAbB2, which has two
identical B subunits and two different A
subunits. When mixed together in the appropriate
stoichiometry, the individual peptides
specifically self-assemble into an asymmetric,
heterotetrameric helical bundle that binds two
metal ions at its active site.
Kaplan, DeGrado, PNAS. 2004, 101, 11566
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A19G and L15A or L15G mutations make a cavity for
the active site
Kaplan, DeGrado, PNAS. 2004, 101, 11566
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kcat 1.3 min-1 KM 0.8 mM kcat/kuncat 1000
Kaplan, DeGrado, PNAS. 2004, 101, 11566
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Our protein shows many of the key features of
biological enzymes in that it has a deeply
invaginated active site into which substrates
bind, and it displays saturation
kinetics. Although the rate is lower than that
observed for many (but not all) highly evolved
enzymes, it nevertheless is significantly greater
than previous de novo-designed proteins and early
catalytic antibodies.
Kaplan, DeGrado, PNAS. 2004, 101, 11566
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Jiang et al., Science 2008, 319, 1387
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Although our results demonstrate that novel
enzyme activities can be designed from scratch
and indicate the catalytic strategies that are
most accessible to nascent enzymes, there is
still a significant gap between the activities of
our designed catalysts and those of naturally
occurring enzymes. Narrowing this gap presents an
exciting prospect for future work What
additional features have to be incorporated into
the design process to achieve catalytic
activities approaching those of naturally
occurring enzymes? The close agreement
between the two crystal structures and the design
models gives credence to our strategy of testing
hypotheses about catalytic mechanisms by
generating and testing the corresponding designs
indeed, almost any idea about catalysis can be
readily tested by incorporation into the
computational design procedure. Determining
what is missing from the current generation of
designs and how it can be incorporated into a
next generation of more active designed catalysts
will be an exciting challenge that should unite
the fields of enzymology and computational
protein design in the years to come.
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